Frozen Breast Tissue Research Articles

Comparison of staining quality between rapid and routine hematoxylin and eosin staining of frozen breast tissue sections: an observational study.

To compare the staining quality between rapid hematoxylin and eosin (H&E) staining and routine H&E staining of frozen breast tissue sections. In this cross-sectional observational study, 120 frozen breast tissue sections were randomly assigned to rapid or routine H&E staining (n = 60 per group). Rapid H&E staining used a 7:1 mixture of modified Gill's hematoxylin and alcohol-soluble 1% eosin Y. The staining quality of each section was evaluated and scored. A score of >7 was considered excellent, a score of 6 to 7 good, and a score of ≤5 poor. The staining time for rapid staining was approximately 3 minutes, whereas that of routine staining was approximately 12 minutes. There were no significant differences in the staining quality scores or proportions of sections in each grade between the two staining methods. The proportions of sections that were classified as excellent or good were 96.7% and 98.3% for rapid and routine staining, respectively. In frozen breast tissue sections, rapid H&E staining may provide staining quality that is comparable to that of routine staining, while markedly reducing the staining time.

Abstract 6333: Quantification of oxylipins as biomarkers of inflammation and cancer risk in breast tissue

Abstract Arachidonic acid (AA) metabolizing enzymes, including cyclooxygenases (COX), lipoxygenases, and cytochrome P450s (CYP450), are expressed in breast cancer (BC). Elevated levels of metabolites of COX-derived, pro-inflammatory prostaglandin (PGs) metabolites in urine have been linked to increased risk of developing breast cancer (BC) in two large prospective cohorts. Further, CYP450-derived EETs in BC tumors are associated with metastasis. We hypothesize that measuring levels of AA-derived oxylipins in breast tissues could enable improved BC risk prediction, provide insights into pro-carcinogenic inflammatory mechanisms, and serve as potential surrogate endpoints in chemoprevention trials. Thus, we performed a pilot study to assess the feasibility of measuring these analytes in small breast tissue samples. We analyzed two sample sets, collected under Institutional Review Board (IRB) approved protocols: 50 normal breast tissue cores donated to the Komen Tissue Bank (KTB) via vacuum-assisted biopsy and 10 tissues removed surgically at Mayo Clinic beyond resection margins of lumpectomy samples performed for atypical ductal hyperplasia (ADH). Tissues were frozen and maintained at -80°C. Normal KTB tissue cores were split in half. Tissues for analysis were weighed, extracted in cold methanol containing a cyclooxygenase inhibitor, and assayed for a panel of 42 oxylipins using validated mass spectrometric methods with a limit of detection per analyte of 0.010 ng/mL in supernatant and CVs of approximately <11%. Analyte measurements in paired cores (ng/g tissue) were compared with Spearman correlations and intra-class correlation coefficients. Measurements in surgical samples were assessed descriptively and levels were compared with normal KTB breast tissues.Median weight of the 50 split KTB cores was 0.055 grams. 17 oxylipins were detected in the tissue samples. PGD2 demonstrated the weakest correlation between split cores (r=0.12; p=0.40), whereas 15-HETE demonstrated the strongest correlation (r=0.68; p<0.0001). ICCs ranged from non-significant to nearly perfect, with highest values for 12-HETE (ICC=0.98, 95%CI: 0.97-0.99). Exploratory analyses of the KTB cores demonstrated significant associations between two BC risk factors, alcohol use and family history of breast/ovarian cancer, and several analytes. Levels of most eicosanoids tested were substantially and highly significantly elevated in tissues surrounding ADH versus KTB normal breast tissues. We conclude that eicosanoids can be readily measured in small frozen breast tissue samples obtained via percutaneous biopsy or at surgery. Future studies to assess the associations of eicosanoid levels with BC risk factors and development of BC after a benign biopsy may inform risk prediction and potentially inhibitable inflammatory mechanisms of breast carcinogenesis. Citation Format: Ginger L. Milne, Robert A. Vierkant, Amy C. Degnim, Anna Maria Storniolo, Jill E. Henry, Laura M. Pacheco-Spann, Derek C. Radisky, Mark E. Sherman. Quantification of oxylipins as biomarkers of inflammation and cancer risk in breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6333.

Abstract P5-08-16: Molecular landscape of the breasts of women at high risk for breast cancer

Abstract Background: Despite advances in breast cancer diagnosis and treatment, there is a critical need to identify the determinants of breast cancer susceptibility in order to define new strategies to target cancer initiation and progression. The Susan G. Komen Tissue Bank at Indiana University (KTB), the only repository worldwide of non-malignant breast samples, can address key scientific questions by examining molecular changes, including epigenetic (DNA methylation) and transcriptomic alterations that occur in breasts of cancer-free women who are considered at high risk for developing breast cancer. Methods: Disease-free breast tissue cores donated by 80 high-risk (Tyrer-Cuzick lifetime risk ≥20%) and 80 average-risk women were processed for whole methylome (Diagenode MeCap Seq Library and NextSeq 75_sequencing) and whole transcriptome (Illumina TruSeq Stranded mRNA HT Library followed by NextSeq 75_sequencing) profiling. The two groups were matched according to age, racial background, and menopausal status. The cohort included 131 pre-menopausal and 29 post-menopausal women (mean age 38.6, SD 12.1). Frozen breast tissue cores with either abundant or moderate epithelial cellularity were selected. For the gene expression analysis, the reads were mapped to the human genome reference, GRCh38.p12 using the alignment software STAR ver. 2.5.2b and the read counts per gene were obtained using featureCounts ver. 1.6.3. For the evaluation of the epigenetically regulated genes, we focused our investigation on two types of functionally relevant epigenetic panels: the first panel includes 666 cancer-related markers (or pan-cancer) described by Shegafinia et al 2018, and the second panel includes 16 epigenetic markers identified in normal breast tissues-derived cells as connected with breast epithelial cell differentiation*. Results: In a preliminary analysis, we searched for transcriptomic differences in the breast tissues of high-risk women (N=43) as compared with that of matched average-risk subjects (N=48).The differential expression analysis identified 126 differentially expressed genes (DEGs, FDR<0.05, fold change >2), including 117 upregulated and 9 downregulated in the high-risk group. Interestingly, pathway analysis (Ingenuity Pathway Analysis, IPA v06_01) showed that most of these genes are involved with cancer. The sub-category “tumorigenesis of tissues” as defined by IPA included 71 genes. Through TCGA database analysis, using the UALCAN portal, we searched among our candidate genes for those targets that showed both overexpression and hypomethylation in breast tumors as compared with the normal breast, or vice versa, downregulation and hypermethylation in breast cancer. This pipeline allowed us to identify 17 genes potentially epigenetically regulated and involved in breast cancer susceptibility and the early phases of cancer development. Among those, two genes, FAM83A and PLA2G3, belong to a family of genes previously described as epigenetically dysregulated in multiple cancers (TCGA dataset, and Shegafinia et al 2018) and transform breast epithelial cell in vitro (FAM83A), thus reaffirming robustness of our approach in identifying genes associated with breast cancer risk Conclusion: The discovery of epigenetically regulated genes associated with breast cancer risk will open the doors to a deeper understanding of the process of cancer initiation and progression. The present study identified 17 gene alterations, two of which are epigenetically regulated in cancer. Our findings highlight an opportunity to address molecular alterations potentially linked with breast cancer susceptibility and risk, using a unique resource of normal breast samples. *Unpublished data by Dr. H. Nakshatri Citation Format: Natascia Marino, Rana German, Harikrishna Nakshatri, Ashley Vode, Jun Liu, Jie Huang, Douglas B Rush, Sha Cao, Anna Maria V Storniolo. Molecular landscape of the breasts of women at high risk for breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-08-16.

Quantitative Dot Blot (QDB) as a universal platform for absolute quantification of tissue biomarkers

To circumvent the limitations associated with sandwich ELISA for tissue biomarker quantitation, Quantitative Dot Blot method (QDB) was proposed using antibodies clinically validated for immunohistochemistry (IHC), as this method requires only one primary antibody in the analysis. The protein levels of four breast cancer tissue biomarkers, including Estrogen Receptor (ER), Progesterone Receptor (PR), Ki67 and Her2, were absolutely quantitated successfully in 190 frozen breast tissue biopsies, and the results were further verified with provided IHC results. We propose QDB method as an alternative platform to Sandwich ELISA for absolute quantitation of tissue biomarkers with significantly reduced developing effort and time.

Rapid evaporative ionisation mass spectrometry of electrosurgical vapours for the identification of breast pathology: towards an intelligent knife for breast cancer surgery

BackgroundRe-operation for positive resection margins following breast-conserving surgery occurs frequently (average = 20–25%), is cost-inefficient, and leads to physical and psychological morbidity. Current margin assessment techniques are slow and labour intensive. Rapid evaporative ionisation mass spectrometry (REIMS) rapidly identifies dissected tissues by determination of tissue structural lipid profiles through on-line chemical analysis of electrosurgical aerosol toward real-time margin assessment.MethodsElectrosurgical aerosol produced from ex-vivo and in-vivo breast samples was aspirated into a mass spectrometer (MS) using a monopolar hand-piece. Tissue identification results obtained by multivariate statistical analysis of MS data were validated by histopathology. Ex-vivo classification models were constructed from a mass spectral database of normal and tumour breast samples. Univariate and tandem MS analysis of significant peaks was conducted to identify biochemical differences between normal and cancerous tissues. An ex-vivo classification model was used in combination with bespoke recognition software, as an intelligent knife (iKnife), to predict the diagnosis for an ex-vivo validation set. Intraoperative REIMS data were acquired during breast surgery and time-synchronized to operative videos.ResultsA classification model using histologically validated spectral data acquired from 932 sampling points in normal tissue and 226 in tumour tissue provided 93.4% sensitivity and 94.9% specificity. Tandem MS identified 63 phospholipids and 6 triglyceride species responsible for 24 spectral differences between tissue types. iKnife recognition accuracy with 260 newly acquired fresh and frozen breast tissue specimens (normal n = 161, tumour n = 99) provided sensitivity of 90.9% and specificity of 98.8%. The ex-vivo and intra-operative method produced visually comparable high intensity spectra. iKnife interpretation of intra-operative electrosurgical vapours, including data acquisition and analysis was possible within a mean of 1.80 seconds (SD ±0.40).ConclusionsThe REIMS method has been optimised for real-time iKnife analysis of heterogeneous breast tissues based on subtle changes in lipid metabolism, and the results suggest spectral analysis is both accurate and rapid. Proof-of-concept data demonstrate the iKnife method is capable of online intraoperative data collection and analysis. Further validation studies are required to determine the accuracy of intra-operative REIMS for oncological margin assessment.

Abstract 2777: One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women

Abstract Altered DNA methylation is an early event in carcinogenesis. Little is known about the mechanism of altered methylation in breast tissue; possible factors include diet such as alcohol and folate intake, and genetic variation for enzymes in one carbon metabolism. Examination of the association of these factors with methylation in breast tissues from healthy women provides insight into these changes. Blood and glandular breast tissues from 81 women with no history of cancer and who underwent reduction mammoplasty were assayed. The 96-plex Illumina BeadXpress® or TaqMan® SNP Genotyping assays assessed SNPs, genome-wide DNA methylation profiling was performed using the Illumina Infinium HumanMethylation450 BeadChip.The Affymetrix GeneChip Human Trascriptome Array 2.0 was used to compare gene expression level with methylation change in fresh frozen breast tissues. Biological networks of differentially-methylated (DM) genes were assigned using the Ingenuity Pathway Analysis (IPA). Fifty-seven CpG sites were DM in comparisons of genotype for eight SNPs in FTHFD, MTHFD1, MTHFR, MTR, MTRR, and TYMS (P<5.0 × 10-5 for each). SNPs in FTHFD were associated with 56% of the DM CpGs. SNPs in FTHFD and MTR were associated with DM CpG sites in their own genes. Six methylation and gene expression pairs were modestly to weakly correlated (P<0.05), five positively correlated (HCN4, FRMD4A, FTHFD, SLC39A7, and LOC63930) and one negatively correlated (ADAMTS14). Four DM CpGs identified by SNPs in MTRR, MTHFR, and FTHFD were significantly associated with alcohol consumption and/or breast folate. Forty-five DM genes were available in the IPA database. IPA revealed enrichment for genes (91%) involved in cancers. The top-scoring network was “Energy Production, Molecular Transportation, Nucleic Acid Metabolism” (score = 32). The top molecular and cellular functions were Amino Acid Metabolism (ALDH1L1, MTR, and PTPRN2), Cell-to-Cell Signaling (DLG3, GRN, HLA-DQB1, PTPRN2, and SLC6A3), Cellular Function and Maintenance (ADAMTS14, ADMTS2, CRIPT, DCL1, DLG3, GRN, KAG2, PTPRN2, and RXRB). High concordance of methylation levels for all DM loci analyzed was found between HM450 and pyrosequencing on 75 technically validated samples (Spearman correlation r = 0.98, P<1.0×10-47). This is the first comprehensive study of the association between variation in one-carbon metabolism genes and genome-wide DNA methylation in histologically normal breast tissues. These SNPs, particularly FTHFD, as well as alcohol intake and folate exposure appear to affect DNA methylation in the breast of healthy women. The finding that SNPs in FTHFD and MTR are associated with their own methylation is also novel and highlights a role for these SNPs as methylation quantitative trait loci. Understanding of the role of one carbon metabolism in altered DNA methylation could provide insight into prevention of breast tumors. Citation Format: Min-Ae Song, Theodore M. Brasky, Catalin Marian, Daniel Y. Weng, Cenny Taslim, Adana A. Llanos, Ramona G. Dumitrescu, Zhenhua Liu, Joel B. Mason, Bhaskar V. Kallakury, Jo L. Freudenheim, Peter G. Shields. One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2777.

Abstract B47: Methylation differences in breast tumor DNA from African American and European women are predominant in estrogen receptor (ER) negative breast cancer and are associated with childbearing

Abstract Background: Although European American (EA) women have higher incidence of breast cancer than African American (AA) women, AA women are more often diagnosed with aggressive estrogen receptor negative (ER-) tumors, the risk of which increases with parity, lack of breastfeeding, and an early age at menarche. DNA methylation could be affected by reproductive factors and may be a potential molecular mechanism driving differences in tumor etiology. In a small study using fresh frozen breast tissue (n=58 AA, 80 EA), we previously found more differentially methylated loci (DMLs) in ER- tumors from AA and EA women than in ER+ tumors. Methods: To follow up on these preliminary findings, we used the Illumina 450K platform to determine genome-wide DNA methylation profiles in formalin fixed paraffin embedded (FFPE) breast tumors from 383 AA and 350 EA women who participated in the Women's Circle of Health Study, a case control study conducted in NY and NJ. We identified DMLs by race and ER status, as well as differentially methylated regions (DMRs). DMLs in eight genes were validated using the Sequenom EpiTYPER platform. Recursively partitioned mixture modeling (RPMM) package in R was used to examine relationships between methylation clusters and reproductive factors, information available from in-person interviews. In addition, using fresh frozen tumor tissue from 50 patients treated at Roswell Park Cancer Institute, we performed RNA sequencing on samples with methylation data available from a prior study, and used Spearmen's correlation to compare methylation and gene expression of DMLs and DMRs. Results: In assessing average methylation by location relative to CpG-islands (CGIs), we found that CGIs and shores in ER- tumors were significantly hypomethylated compared to CGI and shores in ER+ tumors from AA women, but not in tumors from EA women. We also identified 410 DMLs (Δβ>0.10 & FDR<0.05) between AA and EA breast tumors (race-associated DMLs, raDMLs), the majority of which were unique to ER- tumors (n = 260) and hypomethylated in AAs. These loci were enriched for immune response genes and several cell adhesion and inflammatory pathways. RPMM showed that parity, but not breastfeeding or age at menarche, was significantly associated with methylation class of ER- breast tumors. Of the genes that had ER- specific raDMLs, FOXA1 and THSD4 DNA methylation was highly correlated with gene expression (rho=-0.80, rho=+0.87, respectively; pvalue< 2.2x10-16). Summary: ER- tumors from AA women, but not from EA women, exhibited global hypomethylation at CGI and shores compared to ER+ tumors and contained the majority of raDMLs, many of which were associated with genes involved in immune and inflammatory response. The FOXA1 gene, which encodes a pioneer factor previously implicated in suppressing the molecular phenotype of basal breast cancer cells, was found to be methylated and silenced in the majority of ER- tumors analyzed for expression, consistent with aggressiveness of these tumors. These results, and the novel finding of associations between parity and DNA methylation signature, bring us a step forward in understanding the heterogeneous population of ER- tumors and provide insight into mechanisms of racial disparities in breast cancer. Citation Format: Allyson C. Espinal, Dan Wang, Lara Sucheston-Campbell, Song Liu, Qiang Hu, Li Tang, Gary Zirpoli, Thaer Khoury, Song Yao, Kitaw Demissie, Elisa V. Bandera, Christine B. Ambrosone, Michael J. Higgins. Methylation differences in breast tumor DNA from African American and European women are predominant in estrogen receptor (ER) negative breast cancer and are associated with childbearing. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B47.

Abstract 158: Modulation of breast tissue biomarkers by high dose omega-3 fatty acid supplementation in women at high risk for development of breast cancer.

Abstract Background. Previously, we had observed that women at high risk for development of breast cancer were more likely to exhibit cytologic hyperplasia with atypia in specimens acquired by random periareolar fine needle aspiration (RPFNA) if they had low intake and/or low plasma, red blood cell, or breast tissue levels of omega-3 relative to omega-6 fatty acids. We evaluated the effect of high dose omega-3 supplementation on breast tissue markers in two parallel pilot studies, one of pre-menopausal and one of post-menopausal women. Methods. 36 pre-menopausal and 35 post-menopausal women at high risk for breast cancer had breast tissue harvested by RPFNA before and after a 6-month intervention with 4 g daily of omega-3-acid ehyl esters [1.86 g eicosapentaenoic acid (EPA), 1.5 g docosahexaenoic acid (DHA)]. Premenopausal women were aspirated in the follicular phase of the menstrual cycle. Specimens were evaluated for tissue risk biomarkers including cytomorphology, proliferation (Ki-67). Fatty acid composition was determined in plasma, red blood cells, and breast RPFNA specimens. Additional blood and frozen breast tissue was reserved for assessment of hormones, adipokines, cytokines and gene expression. Results. To date, only two of the 71 subjects have discontinued the study early, while 50 subjects have completed study and are evaluable for modulation of tissue biomarkers. Grade 2 or greater gastrointestinal side effects have been reported by only seven subjects. Favorable modulation was observed for cytologic evidence of atypia (70% to 44%; p=0.012), Masood score (medians of 15 to 14; p=0.001), number of epithelial cells recovered (p=0.002), and Ki-67 expression (p=0.059 if all subjects are included even if they did not exhibit Ki-67 staining at baseline, medians of 1.7% to 0.8%; or p=0.001 for 27 women with baseline Ki-67 >1.5%, medians of 3.2% to 1.4%). Modulation was more prevalent (and was statistically significant for all variables) in pre-menopausal women than in post-menopausal women. Fatty acid assessment, adipokine and cytokine assays are batched to minimize variability and all results are not yet available. Preliminary results indicate that the ratio of omega-3:omega-6 fatty acids increased in erythrocytes and plasma by two-fold after 6 months of the high dose omega-3 fatty acid intervention. Conclusion. High dose supplementation with omega-3 fatty acids is well-tolerated in healthy women at high risk for development of breast cancer and was associated with favorable modulation of the tissue risk biomarkers of cytologic atypia and proliferation. This strategy will be explored further as a promising intervention that may reduce risk for development of breast cancer. Supported in part by funding from the Breast Cancer Research Foundation and the Kansas Bioscience Authority. Study agent was provided by GlaxoSmithKline. Citation Format: Carol J. Fabian, Bruce F. Kimler, Carola M. Zalles, Trina Metheny, Jessica A. Box, Jennifer L. Nydegger, Teresa A. Phillips, Brandon H. Hidaka, Susan E. Carlson. Modulation of breast tissue biomarkers by high dose omega-3 fatty acid supplementation in women at high risk for development of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 158. doi:10.1158/1538-7445.AM2013-158

Abstract 1505: Towards an understanding of the different expression profile of fibroblasts from distinct subtypes from breast cancer and normal mammary tissue assessed by transcriptome sequencing.

Abstract The highly heterogeneous clinical, histological, biological and genetic nature of breast malignancies is due in part to their extreme molecular complexity. During breast carcinogenesis, cancer associated fibroblasts (CAFs) have active role in the initiation, progression, metastasis and recurrence of tumors, which adds new levels of complexity to cancer biology, but also brings new prognostic and therapeutic opportunities. We implemented a pilot study to explore the postulation that CAFs within the stroma may exhibit specific gene expression profiles according to the carcinoma subtype. We enrolled frozen breast tissues from nine human breast samples representing three normal tissues, three luminal A and three triple negative subtypes. Fibroblasts were isolated by laser microdissection. For each subtype studied, a cDNA library was constructed and submitted to a transcriptome sequencing (RNA-Seq) on the Genome Sequencer FLX Titanium 454 Plataform. The generated data revealed a total of 14,950 transcripts sequenced and 10,586 genes identified (53.72% of the human genome repertoire). A list of 221 differentially expressed transcripts, obtained by pair wise comparisons among the 3 groups, was ranked by their probabilities of differential expression from 91%-100% (greater expression probability), and from 0.09-9.8% (lower expression probability). Functional similarity interactions (Fun-Net) were constructed using Gene Ontology (GO) annotations as defined in the Entrez Gene database to identify biological functions overrepresented. In addition, 36 differentially expressed genes were selected for technical and biological validation by RT-qPCR (Taqman Low Density Array - TLDA) and tissue microarray (TMA) techniques. Until now 10 out of 20 differentially expressed genes were validated by RT-qPCR. Considering the comparison between fibroblasts associated either to triple negative or luminal breast carcinoma subtypes, five differentially expressed genes were confirmed: S100A13, PPIA, HNRNP0, CUL3 and SLC25A32. Our results indicate that specific changes exist in CAFs derived from breast cancer subtypes, which in turn may contribute to each tumor subtype specific clinical behavior. Supported by FAPESP: 05/60333-7, 09/10088-7. Citation Format: Natália Bromberg, Dirce Maria Carraro, Carlos Alberto Bragança Pereira, Gustavo Campos Molina, Mabel Gigliola Pinilla, Elisa Napolitano Ferreira, Maria Mitzi Brentani. Towards an understanding of the different expression profile of fibroblasts from distinct subtypes from breast cancer and normal mammary tissue assessed by transcriptome sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2013-1505

P5-21-02: The Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center: The Source for Normal Breast Tissue and Biospecimens.

Abstract Background Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. This ignorance has been the consequence of the lack of access to richly annotated, high quality normal breast specimens. The Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB) was established expressly to remedy this deficiency. The KTB is a repository of specimens from volunteer donors with no clinical evidence of breast malignancy. The Bank's mission is to make available specimens that will enable an understanding of the developmental biology of the normal breast, to provide insight into breast oncogenesis, and to provide a normal control for breast cancer research. The purpose of this presentation is to increase the awareness of this unique and rich research resource and to actively solicit the use of its specimens. Methods: The KTB has been prospectively banking fresh frozen breast tissue since mid-2006. Coincident with the tissue donation two tubes of blood are obtained, which are processed for lymphocyte DNA, serum and plasma. These specimens are richly annotated with detailed information regarding the donors’ reproductive history, medical history, family history, and medications. Standard Operating Procedures have been constructed so as to control, limit and identify potential sources of bias. All of this information is recorded in an Oracle-based, searchable database. Results: As of June 2011, the KTB and its predecessor bank, Mary Ellen's Bank, have available fresh frozen breast tissue (10 gauge cores) from 1469 donors; formalin-fixed, paraffin-embedded tissue from 1055; DNA from 7507; serum from 2382; and plasma from 3771 donors. The KTB has also established 28 epithelial and 33 stromal cell lines from the cores; 4 of the epithelial cell lines have been immortalized using hTERT. Donors range in age from 18–86 years of age. 9% of donors to the KTB describe themselves as Hispanic/Latino. 5.2% of donors are Black or African-American. Using the Gail Risk Model, there is a bimodal distribution of life-time breast cancer risk among the donors: the largest peak is at 10% and a smaller one at 18%. Conclusions: The KTB is a unique and invaluable research resource which is now open for business and accessible to researchers across the globe. We encourage researchers to avail themselves of this unique tissue resource and to also acquaint themselves with other sources of healthy breast tissue, i.e., the Love/Avon Army of Women [http://www.armyofwomen.org/]. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-21-02.

Abstract LB-180: Epigenetic portraits of human breast cancers

Abstract Background: Understanding the diversity of breast cancer is essential to improving diagnosis and optimising treatment. Both genetic and acquired epigenetic abnormalities participate in cancer, but information is scant on the involvement of the epigenome in breast cancer and its contribution to the complexity of the disease. Our goal was to explore the DNA methylation landscapes of phenotypically heterogeneous tumours, to relate this diversity to landscape features, and extract biological and clinical meaningful information. Methods: We performed comprehensive DNA methylation profiling to assess the methylomes of two independent sets of frozen breast tissue samples: a “main set” of 123 samples (4 normal and 119 infiltrating ductal carcinomas, IDCs), and a “validation set” of 125 samples (8 normal and 117 IDCs). We used the recently developed Illumina's Infinium Methylation Assay, that allows to assess the methylation status of more than 27,000 CpGs corresponding to over 14,000 genes. Results: Firstly, it emerged that the two major phenotypes of breast cancers determined by ER status are widely epigenetically controlled. Secondly, we have distinguished, and validated in an independent set of tumours, 6 methylation-profile-based tumour groups, some coinciding with known “expression subtypes” but also new entities that may provide a meaningful basis for refining breast tumour taxonomy. Thirdly, we showed that DNA methylation profiling can reflect the cell type composition of the tumour microenvironment. Lastly, we highlighted an unexpectedly strong epigenetic component in the regulation of key immune pathways, revealing a set of immune genes having high prognostic value in specific tumour categories. Conclusions: In this study, we have generated the largest and most comprehensive DNA methylation data set for human breast tumor tissues. Several novel findings and original concepts for breast cancer emerge, that previous RNA expression profiling has not highlighted. By laying the ground for better understanding of breast cancer heterogeneity and improved tumor taxonomy, the precise epigenetic portraits drawn in our work should contribute to better management of breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-180. doi:10.1158/1538-7445.AM2011-LB-180

The Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center: The Source for Normal Breast Tissue and Biospecimens.

Abstract Background: Our efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. This ignorance has been the consequence of the lack of access to richly annotated, high quality normal breast specimens. To provide the specimens that will enable the study of normal mammary development and to provide normal controls for breast cancer research, the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is a repository of specimens from volunteer donors with no clinical evidence of breast malignancy. The purpose of this presentation is to increase the awareness of this unique and rich research resource and to actively solicit the use of its specimens.Methods: The initial collection venue was the 2005 Komen Indianapolis Race for the Cure® at which whole blood was collected, and processed for DNA. The collection of serum began one year later. The KTB has been prospectively banking fresh frozen breast tissue since mid-2006. Plasma processing began in 2008. These specimens are richly annotated with detailed information regarding the donors' reproductive history, medical history, family history, and medications. Standard Operating Procedures have been constructed so as to control, limit and identify potential sources of bias. All of this information is recorded in an Oracle-based, searchable database.There are five, straight-forward steps to submit a proposal for specimen access: 1. Generate a hypothesis and design a study to test the hypothesis; 2. Perform a preliminary statistical analysis to determine the number of samples needed to test the hypothesis; 3. Access the Komen Tissue Bank on-line and determine if the Bank has the type and number of specimens to fulfill your research needs. A sample/data search can be performed on-line at https://komentissuebank.iu.edu/search; 4. Obtain IRB approval from your institution and secure funding for your research project; 5. Submit your proposal to the KTB using the on-line form. Deadlines are February 1, June 15 and October 15 yearly. Proposals are reviewed by the Proposal Review Committee, composed of internal and external scientific experts and patient advocates. Additional information can be found on the KTB website: https://komentissuebank.iu.edu/research.Results: As of June 2009, the KTB and its predecessor bank, Mary Ellen's Bank, have available fresh frozen breast tissue (10 gauge cores) from 508 individual donors, DNA from 2524, serum from 1360 and plasma from 1438 donors.Figure 1 Life-time risk of the development of breast cancer calculated using the Gail Model for tissue donors to the KTB. x-axis: % lifetime risk, y-axis number of tissue donors, each line =10Conclusions: The KTB is a unique and invaluable research resource which is now open for business and accessible to researchers across the globe. We encourage researchers to avail themselves of this unique tissue resource and to also acquaint themselves with other sources of healthy breast tissue, i.e., the Love/Avon Army of Women [http://www.armyofwomen.org/]. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3076.

Expression of MDA7/IL-24 human breast cancer and the molecular impact.

Abstract Abstract #2060 INTRODUCTION. MDA-7 (melanoma differentiation associated gene-7), also known as IL-24, was initially identified from cancer cells and was found to be up-regulated in melanoma cells. Forced expression of MDA7 in cancer cells was found to be growth inhibitory. MDA7/IL-24 operates in cells via its receptor, MDA7R/IL-24R, which includes at least the IL-20Rα and IL-20Rβ complex and the IL-22R and IL20Rβ complex. The present study investigated the impact of MDA7 on the growth and motility of breast cancer cells and the clinical relevance of MDA7 expression in mammary tumour tissues.
 MATERIALS AND METHODS. Human breast cancer cell lines MDA MB-231 and MCF-7 were used for in vitro testing. The response of cancer cells to rhMDA7 in growth and cellular motility was determined using growth assay and ECIS (electric cell impedance sensing, and confirmed by wounding assay). Localisation of MDA7 in mammary issues was assessed using conventional immunohistochemical method. Levels of MDA7 transcript in fresh frozen breast tissues were determined using quantitative PCR analysis and analysed against clinical and pathological information.
 RESULTS. Recombinant human MDA-7 only had a marginal effect on the in vitro growth of the breast cancer cell lines used. However, rhMDA had a significant effect on the migration of breast cancer cells. Cells treated with rhMDA-7 showed a slower rate of migration (electrical resistance 80.1±24.3Ω), when compared with control cells (130±24.3Ω) (p=0.024). Normal mammary epithelial cells showed a good degree of staining of MDA-7, a staining greatly reduced in cancer cells. Using the Nottingham Prognostic Index as a predictor of prognosis, tumours from patients with poor prognosis showed a significantly lower level of MDA-7 transcript compared with tumours from those with good prognosis (p=0.049). A significant difference was seen between tumours from patients who died of breast cancer and tumours from patients who remained disease free (p=0.035), the later showing a higher levels of MDA7 transcript. Using the Kaplan-Meier survival analysis, it was revealed that low levels of MDA-7 were significantly correlated with a shorter disease free survival (mean survival for low level group 121.7 (108.5-134.9) months compared with high levels 140.4 (133.7 – 147.1, 95% CI) months, p=0.0287. ER positive tumours showed a lower but levels of MDA-7 expression compared with ER-negative tumours, although this was not statistically significant (p=0.094).
 CONCLUSIONS. MDA-7 is a potential regulator of cellular motility of breast cancer cells. The aberrant expression of the molecule is linked to the prognosis and long term survival of patients, indicating the potential clinical implication of MDA-7 in cancer therapies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2060.

Cyclo-oxygenase-2 (COX-2) mRNA expression correlates with progesterone receptor positivity in human breast cancer.

We previously presented evidence showing that cyclo-oxygenase 2 (COX-2) plays an important role in mammary carcinogenesis and angiogenesis in human breast cancer. The present study aims to compare COX-2 mRNA expression with hormone receptor status, S-phase fraction, telomerase activity, and DNA ploidy in human breast cancer. Total cellular RNA was extracted from frozen breast tissue samples according to standard methodology. The mRNA copy numbers for COX-2 were determined in 18 infiltrating carcinomas using quantitative RT-PCR and TaqMan methodology. The oestrogen receptor (ER) and progesterone receptor (PgR) status was determined using the ligand-binding technique (ER+ = > 3 fmol/mg, PgR+ = > 5 fmol/mg). We also determined DNA ploidy status (diploid or aneuploid), S-phase fraction (< 6% = low, 6-10% = intermediate, > 10% = high), and telomerase activity (total protein generated by TRAP assay). The median COX-2 mRNA copy number per micro g of RNA was 126 713 (range = 15 717-2 022 050). COX-2 expression was significantly associated with PgR positivity (p = 0.013). The association between COX-2 and DNA diploidy failed to reach a statistical significance (p = 0.085). No significant association was detected between COX-2 and S-phase fraction, ER status, or telomerase activity. COX-2 mRNA expression is associated with PgR positivity in human breast cancer. This observation is consistent with the hypothesis that COX-2 upregulates aromatase activity.

Merging of orthogonally sampled projection sets as a means for eliminating correlation artifacts from tomograms

We explore the feasibility of merging independent data sets to mitigate volume anisotropy intrinsic to tomosynthetic reconstructions. Two independent sets of orthogonally oriented projection data are obtained, respectively, from a hand phantom and from frozen breast tissues. Both objects are enclosed within radiolucent containers containing multiple fiducial reference objects. The latter facilitates registration of multiple projections produced by incrementally moving the x-ray source relative to the object about a single axis through a fixed series of angles. These data encompass maximum angular disparities up to 90 deg for each projection series. The resulting data are projectively transformed and nonlinearly processed using tuned-aperture computed tomography to yield a number of contiguous slices equal to the linear resolution of the sampled projections measured in pixels. The resulting slice data are then corrected for differential magnification, appropriately rotated, and linearly merged to yield a relatively complete, volumetrically isotropic representation of the phantom that could be visualized from any desired angle with negligible apparent tomosynthetic distortion. The resulting displays are evaluated subjectively and compared quantitatively with control images produced from optimum projection geometries. The results are consistent with the hypothesis that volume anisotropy intrinsic to tomosynthetic reconstructions can be minimized through integration of contiguously sampled orthogonal projections.

Cyclo-oxygenase-2 (COX-2) mRNA expression correlates with progesterone receptor positivity in human breast cancer

SUMMARYBackground: We previously presented evidence showing that cyclo-oxygenase 2 (COX-2) plays an important role in mammary carcinogenesis and angiogenesis in human breast cancer. The present study aims to compare COX-2 mRNA expression with hormone receptor status, S-phase fraction, telomerase activity, and DNA ploidy in human breast cancer.Methods: Total cellular RNA was extracted from frozen breast tissue samples according to standard methodology. The mRNA copy numbers for COX-2 were determined in 18 infiltrating carcinomas using quantitative RT-PCR and TaqMan methodology. The oestrogen receptor (ER) and progesterone receptor (PgR) status was determined using the ligand-binding technique (ER+ = >3 fmol/mg, PgR+ = >5fmol/mg). We also determined DNA ploidy status (diploid or aneuploid), S-phase fraction (<6% = low, 6-10% = intermediate, > 10% = high), and telomerase activity (total protein generated by TRAP assay).Results: The median COX-2 mRNA copy number per ug of RNA was 126 713 (range = 15717-2022050). COX-2 expression was significantly associated with PgR positivity (p = 0.013). The association between COX-2 and DNA diploidy failed to reach a statistical significance (p = 0.085). No significant association was detected between COX-2 and S-phase fraction, ER status, or telomerase activity.Conclusions: COX-2 mRNA expression is associated with PgR positivity in human breast cancer. This observation is consistent with the hypothesis that COX-2 upregulates aromatase activity.

The mRNA Expression of Cyclo-oxygenase-2 (COX-2) and Vascular Endothelial Growth Factor (VEGF) in Human Breast Cancer

SummaryAims: There is a growing body of evidence that cyclo-oxygenase 2 (COX-2) plays an important role in carcinogenesis and angiogenesis of human tumours. The present study aims to compare COX-2 expression in human breast cancer and adjacent non-cancerous tissue (ANCT), and to identify any correlation between COX-2 and VEGF expression.Methods: Total cellular RNA was extracted from frozen breast tissue samples according to standard methodology. The mRNA copy numbers for COX-2 and vascular endothelial growth factor 189 (VEGF-189) were determined 40 infiltrating carcinomas and 40 matched ANCT specimens using quantitative RT-PCR and TaqMan methodology.Results: The COX-2 mRNA copy number per ixg of RNA was two-fold higher in ANCT compared with the cancerous tissue (p = 0.01). Median mRNA copy number was 5.44 x 106 for ANCT and 2.30 x 106for tumour, (ANCT range: 1 x 106to4.12x 107) (tumour range: 1.29 x 105to1.07x 107).There was a significant correlation between COX-2 and VEGF-189 mRNA copy numbers in the cancer specimens (correlation coefficient = 0.5528, p = 0.0076).Conclusions: COX-2 mRNA is overexpressed in both human breast cancer and ANCT. We found higher levels in the matched ANCT which suggests that paracrine effects may be important in the role of COX-2 in mammary carcinogenesis. Furthermore, our results indicate that in human breast cancers COX-2 overexpression is linked to VEGF-189 overexpression and therefore tumour angiogenesis.